Imaging with the highest possible resolution of individual regions of interest in a series of slices is desirable, particularly for biological samples. Three-dimensional information, in particular, in respect of specific regions of interest within a sample volume is often obtained in this fashion.
To this end, a sample is usually cut into a multiplicity of, often up to several hundred, thin slices. The slices are arranged on suitable sample carriers and are initially observed, particularly via light or electron microscopy, and regions of interest sought after. A detected selected region of interest is subsequently directed into the center of the field of view and thereafter targeted and imaged with high magnification. Corresponding regions on the neighboring slices (neighboring regions) are now sought after manually with great difficulty and these regions are subsequently likewise imaged at high magnification (“conventional approach” in Vicidomini et al., High Data Output and Automated 3D Correlative Light-Electron Microscopy Method, Traffic 2008; 9: 1828-1838). The results of this complicated process are very often very unsatisfactory and results which are at all utilizable can only be obtained with much experience and skill. In the case of optical microscopes, there is the basic problem in that light-sensitive preparations very often already experience irreversible damage in the time required for finding corresponding regions again and for example bleach before the individual regions of interest and the neighboring regions thereof are captured in image form with the desired quality. Comparable problems occur in the particle beam microscope. By way of example, a sample in the electron microscope is, under certain circumstances, badly damaged and contaminated by a continuous bombardment with electrons. In the scanning electron microscope in particular, the dwell period at individual sample regions should be kept as short as possible because otherwise there is a risk of the sample charging locally, which prevents satisfactory imaging qualities.
Various efforts have been undertaken to optimize the imaging process. By way of example, US2008/0152207 A1 describes a method according to which the slices of a sample are sought after and captured in overview images via an image recording apparatus. The search for regions of interest then does not occur in the specimen, but retrospectively on the image thereof. A disadvantage of the method is that, in terms of their lateral resolution, the individual regions of interest are restricted to the resolution at which the overview images of the individual slices were created. Vicidomini et al (Vicidomini et al., High Data Output and Automated 3D Correlative Light-Electron Microscopy Method, Traffic 2008; 9: 1828-1838) describes a method which addresses this problem. According to this, the sample is likewise initially captured in image form via the thinnest individual images and the individual images are subsequently assembled to form a higher-resolution mosaic image. In this mosaic image, the image information corresponding to the regions of greatest interest is subsequently extracted from the mosaic image in a manual process and the image stack is then reconstructed to form a three-dimensional volume, i.e. an image data volume. Although this approach achieves an increased lateral resolution, the manual search within the individual slices for corresponding regions remains extremely complicated in the case of up to several hundred slices per sample. As a result of the long recording times, this method moreover takes a very long time and generates huge amounts of data.